nutrients Article Explorative Screening of Bioactivities Generated by Plant-Based Proteins after In Vitro Static Gastrointestinal Digestion Camille Dugardin 1, Benoit Cudennec 1,* ,Mélissa Tourret 1, Juliette Caron 2, Laetitia Guérin-Deremaux 2, Josette Behra-Miellet 1, Catherine Lefranc-Millot 3 and Rozenn Ravallec 1,* 1 UMR-T 1158, BioEcoAgro, University of Lille, F-59000 Lille, France; [email protected] (C.D.); [email protected] (M.T.); [email protected] (J.B.-M.) 2 Nutrition and Health Research & Development, Roquette, F-62136 Lestrem, France; [email protected] (J.C.); [email protected] (L.G.-D.) 3 Nutrition and Health Research & Development, Roquette, F-59110 La Madeleine, France; [email protected] * Correspondence: [email protected] (B.C.); [email protected] (R.R.) Received: 13 October 2020; Accepted: 2 December 2020; Published: 5 December 2020 Abstract: The gastrointestinal digestion of food proteins can generate peptides with a wide range of biological activities. In this study, we screened various potential bioactivities generated by plant-based proteins. Whey protein as an animal protein reference, five grades of pea protein, two grades of wheat protein, and potato, fava bean, and oat proteins were submitted to in vitro SGID. They were then tested in vitro for several bioactivities including measures on: (1) energy homeostasis through their ability to modulate intestinal hormone secretion, to inhibit DPP-IV activity, and to interact with opioid receptors; (2) anti-hypertensive properties through their ability to inhibit ACE activity; (3) anti-inflammatory properties in Caco-2 cells; (4) antioxidant properties through their ability to inhibit production of reactive oxygen species (ROS). Protein intestinal digestions were able to stimulate intestinal hormone secretion by enteroendocrine cells, to inhibit DPP-IV and ACE activities, to bind opioid receptors, and surprisingly, to decrease production of ROS. Neither pro- nor anti-inflammatory effects have been highlighted and some proteins lost their pro-inflammatory potential after digestion. The best candidates were pea, potato, and fava bean proteins. Keywords: plant-based protein digestion; biological activity; CCK; GLP-1; DPP-IV; opioid receptor; ACE; inflammation; IL-8; ROS production 1. Introduction According to the Food and Agriculture Organization (FAO), current trends predict a constant increase in worldwide population, reaching nearly 10 billion people in 2050. Moreover, the international recommended dietary allowance for protein is currently 0.8 g per kg of body weight [1]. As a consequence, global protein demand should increase to meet the nutritional needs of the growing population. Additionally, since 1961, worldwide protein consumption has increased by about 20 g per day and per person. In this context, it becomes essential to better characterize dietary proteins and to diversify their origins. Whereas the high proportion of animal-protein consumption in developed countries raises environmental concerns, relying on intensive livestock farming [2], plant-based proteins appear as a more sustainable alternative. Indeed, the environmental footprint of plant-based proteins is lower due to less greenhouse gas emission and water consumption, leading to a better protein delivery efficiency [3]. Moreover, the proportion of the population becoming vegan, vegetarian, Nutrients 2020, 12, 3746; doi:10.3390/nu12123746 www.mdpi.com/journal/nutrients Nutrients 2020, 12, 3746 2 of 19 or flexitarian is increasing, promoted by ecological and health concerns but also the ethical treatment of animals [4]. In parallel, according to the World Health Organization, more than 1.9 billion adults were overweight in 2016. Among them, more than 600 million were obese (13% of the worldwide adult population). Obesity is a major risk factor for the development of type 2 diabetes mellitus (T2DM), which is a metabolic disorder characterized by prolonged hyperglycemia, leading to complications like hypertension and associated cardiovascular diseases. Beyond their nutritional role as the source of amino acids for protein synthesis, dietary proteins are known to be involved in a wide range of biological functions [5], particularly through the action of peptides generated during their digestion in the gastrointestinal tract. Indeed, during this enzymatic process, dietary proteins are first partially hydrolyzed by pepsin in the stomach, then by a cocktail of proteases (trypsin, chymotrypsin, carboxypeptidases) in the small intestine, and finally, by peptidases at the brush border membrane. This generates amino acids and bioactive peptides present in the intestinal lumen whose size, sequence, and structure vary and which modulate several physiological processes by acting locally and/or systemically [6–8]. Several health-related effects have been described for bioactive peptides coming from different sources, using in vitro tests or animal and human trials [9,10]. For instance, it has been shown that peptides generated by dietary protein digestion could play a beneficial role in the context of obesity and metabolic disorders through the peripheral regulation of food intake [11]. Peripheral regulation of short-term food intake by proteins involves the stimulation of intestinal hormone secretion following the recognition of nutrients, such as digested protein-derived peptides, at the apical level of the enteroendocrine cells. This intestinal “sensing” leads to the secretion of anorexigenic peptide hormones such as cholecystokinin (CCK) and glucagon-like peptide 1 (GLP-1) that act as peripheral signals leading to the end of food intake [12,13]. GLP-1 plays also a crucial role in glucose metabolism by its role as incretin [14], which is drastically reduced by the dipeptidyl peptidase-4 (DPP-IV) enzymatic action, removing dipeptides from their N-terminal side [15]. Hence, DPP-IV inhibitors are nowadays considered as an advanced class of agents for T2DM management [16] and in recent years, numerous studies evidenced “natural” DPP-IV inhibitory peptides from digested dietary proteins [17]. Food-derived peptides may also bind peripheral opioid receptors in the portal vein and indirectly induce satiety via gluconeogenesis [18,19]. Dietary protein-derived peptides are also promising in the context of hypertension and associated cardiovascular risk, particularly by their ability to inhibit the Angiotensin Converting Enzyme (ACE), a dipeptidyl carboxypeptidase which plays an important role in the regulation of blood pressure by cleaving angiotensin I to induce vasoconstriction [20]. Several in vitro and animal trials had also evidenced the attractive potential of food-derived peptides in the management of human inflammatory bowel disease (IBD) via their anti-inflammatory effect and antioxidant activities [21]. However, data describing the relationship existing between the source and quality of protein and their biological activities are limited and inconsistent. Moreover, studies comparing numerous protein sources are lacking. The purpose of the present study was thus to investigate in vitro the ability of 10 dietary plant-based proteins to modulate (1) food intake and glucose homeostasis through CCK and GLP-1 secretion, DPP-IV activity, and opioid receptor binding; (2) blood pressure through ACE activity; (3) inflammation through interleukin-8 (IL-8) secretion; (4) oxidative stress through reactive oxygen species (ROS) production. To do that, an in vitro simulated gastrointestinal digestion (SGID) was firstly performed in order to reach study conditions closer to the physiological ones but also to highlight and compare the effect of non-digested and digested dietary proteins on these biological activities. The protein sources have been chosen for two reasons. On the one hand, some of these sources are rich in proteins (e.g., pea) and thus, interesting for human consumption. On the other hand, some of these sources generate a lot of protein by-products (e.g., potato), which could be valorized. Nutrients 2020, 12, 3746 3 of 19 2. Materials and Methods 2.1. Protein Samples Ten plant-based protein samples were provided by Roquette (Table1): - Four grades of pea protein (PeaP1, PeaP2, PeaP3, and PeaP4); - Hydrolyzed pea protein (HPeaP); - Two grades of wheat protein (WP1 and WP2); - Potato protein (PP); - Fava bean protein (FBP); - Oat protein (OP). Table 1. Description and characterization of the 11 protein samples studied. Name Description % Dry Matter % Protein PeaP1 Pea protein—grade 1 93.0 79.1 PeaP2 Pea protein—grade 2 95.2 79.7 PeaP3 Pea protein—grade 3 94.4 80.8 PeaP4 Pea protein—grade 4 95.6 81.5 HPeaP Hydrolyzed pea protein 94.6 78.3 WP1 Wheat protein—grade 1 94.8 83.7 WP2 Wheat protein—grade 2 93.2 93.2 PP Potato protein 93.8 79.1 FBP Fava bean protein 96.0 89.8 OP Oat protein 96.0 88.3 WhP Whey protein 95.0 85.5 PeaP1–PeaP4 (4 grades of pea protein), HPeaP (hydrolyzed pea protein), WP1–WP2 (2 grades of wheat protein), PP (potato protein), FBP (fava bean protein), OP (oat protein) and WhP (Whey protein). We performed two sets of analysis: the first one including PeaP1, HPeaP, WP1, WP2, and PP; the second one including PeaP2, PeaP3, PeaP4, FBP, and OP. Whey protein (WhP) was used as an animal protein reference and common control in each set of analysis. 2.2. Materials 1 Porcine pepsin (EC 3.4.23.1, from porcine gastric mucosa, 3850 U mg− protein), pancreatin from porcine pancreas (4 USP specifications (5.46 U mg 1 based on trypsin activity), EC 232-468-9), × − Dipeptidyl Peptidase IV (DPP-IV from porcine kidney, EC 3.4.14.5, 10 U mg 1 protein), ≥ − Gly-Pro-p-nitroanilide hydrochloride, Angiotensin Converting Enzyme (ACE from rabbit lung, EC 3.4.15.1), and all other reagents were purchased from Sigma-Aldrich (Sigma-Aldrich, Steinheim, Germany). The Active Glucagon-Like Peptide RIA kit (Cat.# GLP1A-35HK) was purchased from Merck Millipore (Merck-Millipore, Darmstadt, Germany). The Gastrin/CCK kit was purchased from CisBio (CisBio, Saclay, France). Human IL-8/CXCL8 Quantikine ELISA Kit (D8000C) was purchased from R&D Systems (R&D Sytems, Minneapolis, MN, USA).